In order to identify the cellular proteins that are associated with neoplastic development, we have used an in vitro transforming system consisting of helper-dependent retroviruses containing either v-raf or v-raf/v-myc as the transforming agents and rat liver epithelial (RLE) cell line as the reporter cell. Both v-raf (3611) and v-raf/v-myc (J2) viruses were efficient in vitro transforming agents of the RLE cells and the infected cells formed tumors upon transplantation in nude mice. Neither the uninfected nor helper virus-infected RLE cells formed tumors in nude mice. Cellular protein patterns in normal RLE cells, helper-infected (RLE-H), 3611 (RLE-3611T)- and J2 (RLE-J2)-transformed RLE cells have been analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Comparison of the autoradiograms from the RLE-H, RLE-3611T and RLE-J2, nine proteins which are either suppressed or induced were selected for closer study and purification. Polypeptides 1 - 4 (Mr/pI) (1. 145/5.9-7.2; 2. 130/6.5-7.5; 3. 100/4.0; 4. 30/6.0) were found be present in negligible amounts in the tumorigenic cells from both autoradiograms and silver-stained 2-dimensional polyacrylamide gels and polypeptides 5 - 9 (Mr/pI) (5. 27/5.0; 6. 26/4.9; 7. 25/4.9; 8. 25/5.2; 9. 27/6.6) were found to be present in negligible amounts in the nontumorigenic RLE-H cells. Protein 1 appears to be associated with both soluble and membrane fractions; polypeptides 2, 3, 4, 8 and 9 appear to be associated with membrane and polypeptides 5, 6 and 7 appear in the cytosolic fraction.